The invention relates to a method of chromatography and in particular to temperature related affinity chromatography.
Affinity chromatography is a widely used technique to purify proteins. This technique makes use of specific binding between two species for example two proteins. A sample containing a protein to be isolated is applied to a solid support having the other protein bound thereto. The specificity of binding between the two proteins leads to highly selective binding of the protein to be isolated to the solid support. A particular example is immunoaffinity chromatography where anti-ligand antibodies are bound to a solid support for purification of the ligand from a sample.
For affinity chromatography, it is desirable that the support bound protein has a strong and specific affinity for a desired protein to be purified. This strong and specific affinity is used to ensure that a high proportion of the desired protein in the sample is bound to the column with little or no binding of other impurities in the sample.
The desired protein must then be removed from the column. It is desirable that this protein will separate easily from the support bound protein so that the desired protein can be recovered in high yield and without affecting its integrity. However these conditions are often in conflict with the ability of the support-bound protein to bind the desired protein with high affinity and specificity. Thus in some circumstances the elution conditions required to remove the desired protein are unduly harsh and may lead to some denaturing or degradation of protein. Examples of current elution techniques are affinity elution with substrate or free ligand, change in pH or ionic strength, addition of a denaturing agent or changes in solvent polarity.
This technique can also be used to remove specific proteins from a sample e.g. immunoglobulins from plasma. Although in these circumstances there may be no further use for the immunoglobulin, it may still be desirable to release immunoglobulin from the column so that the column may be reused. As outlined above, the conditions required to elute the proteins from the column may be harsh, affecting not only immunoglobulin but also support bound protein.
This invention provides a method of affinity chromatography which makes use of a simple change in temperature to facilitate elution of a protein from a solid support thus avoiding harsh changes required in the prior art techniques. A coiled coil protein which is capable of forming a homodimer which dissociates into monomers on change of temperature is used in the method of isolating a ligand or desired protein from a sample.
In a first aspect the invention provides a method of isolating a ligand comprising providing a solid support having bound thereto a coiled coil protein which binds to said ligand and whose affinity for the ligand is temperature dependent, contacting a sample containing the ligand with the support at a temperature which promotes binding of the ligand to the protein and subsequently altering the temperature to promote dissociation of the ligand from the protein.
In a second aspect the invention provides a method of isolating a desired protein comprising tagging said protein with a coiled coil protein whose affinity for a ligand is temperature dependent or with a ligand which binds to the coiled coil protein; providing a solid support having bound thereto the other of the ligand or coiled coil protein; contacting a sample containing the tagged protein with the support at a temperature which promotes binding between the coiled coil protein and ligand; and subsequently altering the temperature to promote dissociation of the ligand from protein.
In a third aspect the invention provides a method of isolating a desired protein comprising tagging said protein with a coiled coil protein monomer; providing a solid support having bound thereto coiled coil protein monomer; contacting a sample containing the tagged protein with the support at a temperature which promotes binding between the monomer-tagged protein and support bound monomer to form a coiled coil; and subsequently altering the temperature to promote dissociation of the desired protein-monomer from the support bound monomer.
The temperature is generally raised to promote dissociation of binding.